CN1110567C

CN1110567C - Preparation of deoxynucleoside triphosphate

Info

Publication number: CN1110567C
Application number: CN00100844A
Authority: CN
Inventors: 郭焕芳
Original assignee: BEIJING TAIGONG BIOLOGICAL TECHNOLOGY Co Ltd
Current assignee: Beijing Taigong Biological Technology Co., Ltd.
Priority date: 2000-02-17
Filing date: 2000-02-17
Publication date: 2003-06-04
Anticipated expiration: 2020-02-17
Also published as: CN1269422A

Abstract

The present invention relates to a method for preparing requisite precursor raw materials for artificially synthesizing DNA. The present invention adopts the novel technology of enzymolysis-enzyme promotion-chemosynthesis. DNMP can be obtained by the enzymolysis of natural DNA by deoxyribonuclease in a solution state, and a DNMP yield is between 70 and 90%. The DNMP can be respectively converted into corresponding DNTP by one-step chemical reaction after the enzyme reaction of the DNMP. DNTP with the purity of 95% to 97% can be obtained by chromatography purification and the precipitation and the separation in the form of sodium salts or barium salts. The production technology of DNTP becomes simple by the present invention, and the production quantity achieves a molar scale. The present invention is suitable for industrial production, and has little pollution.

Description

The preparation method of deoxyribonucleoside triphosphate

The present invention relates to artificial-synthetic DNA's technology, particularly relate to the method for artificial-synthetic DNA's indispensable precursor raw material.

As everyone knows, deoxyribonucleoside triphosphate (DNTP) is artificial-synthetic DNA's an indispensable precursor raw material, and according to the difference of its structure, DNTP can be divided into DATP, DCTP, DGTP and DTTP.Artificial-synthetic DNA's segment (gene) is widely used in aspects such as genetically engineered, molecular biology, life science, genomic medicine.The preparation research of DNTP once had many reports, as Chemica1 Abstract88,117297; ChemicalAbstract89,180281; Acta.Biochim.biophys.Acad.Sci.Hung.16,131-3 (1981).But all these methods all have certain defective, and the resultant quantity of most biological production is below 1mmol, and the synthetic aftertreatment that has of chemical method is difficulty quite, the organic solvent that also needing of having uses a large amount of toxicity bigger.

The object of the present invention is to provide a kind of method of utilizing enzymolysis-enzymatic-chemical synthesis process to prepare deoxyribonucleoside triphosphate.

The preparation method of deoxyribonucleoside triphosphate of the present invention comprises that the following step poly-:

The substratum of DNMP enzyme preparation is formed: microbial culture yeast extract 5-10%, peptone 0.5-2%, potassium primary phosphate 4-6%, dipotassium hydrogen phosphate 2-5%, glucose 10-15%, urea 0.5-3%, calcium chloride 0.2%, zinc chloride 0.2%, substratum is after the steam sterilizing sterilization of 1.2 kg/cm, inoculation, inoculum size is 20-30%, 50 ℃ of following violent stirring were cultivated 10-30 hour, centrifugal collection thalline under 1 ℃; Thalline is after high-speed stirring, damping fluid extraction with PH2-7, damping fluid can be used Trisodium Citrate-sodium hydroxide-hydrochloric acid, Repone K-hydrochloric acid, acetate-sodium acetate, citric acid-sodium citrate, Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic-potassium primary phosphate, potassium primary phosphate-sodium hydroxide, Sodium phosphate dibasic-citric acid, Potassium Hydrogen Phthalate-sodium hydroxide etc., and extraction liquid is not purified can directly to be used.

Commodity DNA is dispersed in and makes aqueous dna in the water, and DNA can come from whale essence, salmon essence, carp essence, beef liver, ox thymus gland etc. respectively.Adding zine ion in dna solution makes its concentration reach 2 * 10 ^-2-1 * 10 ^-4M, zine ion can be provided by zinc acetate, zinc chloride, zinc sulfate etc.Activity according to deoxyribonuclease adds its amount, is generally 20 of dna solution amount--and 1/50th.Enzymolysis carries out under 30-55 ℃; Enzymolysis 5-15 hour.

It is 4-8 that the DNMP aqueous solution is regulated PH with the potassium hydroxide solution of 10M, adds phosphoric acid salt, as Disodium phosphocreatine, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, PEP etc.; Add metal ion again, as zinc sulfate, zinc chloride, zinc acetate, calcium chloride, sal epsom, magnesium chloride, bariumchloride, make catalyzer with ATP, the activity adding DNMP enzyme according to the DNMP enzyme is generally every mmole DNMP and adds 40-60 unit's enzyme.50-55 ℃ is incubated 48-96 hour.

After enzymatic reaction is finished, add the alcoholic solvent of equivalent, as methyl alcohol, ethanol, butanols, butyl cellosolve, the DCC that phosphoric acid that 2-5 doubly measures or phosphoric acid salt, 20-40 are doubly measured, 80-90 ℃ was reacted 4-8 hour down.

DNTP is synthetic finish after, the solution that obtains is phosphoric acid salt, the mixing solutions of DNMP, DNDP, DNTP and polyphosphide, this solution can obtain purified DNTP through chromatographic separation.

The present invention adopts the brand-new technology circuit of enzymolysis-enzymatic-chemosynthesis, adopts n DNA can obtain DNMP through the enzymolysis of solution state deoxyribonuclease, and the yield of DNMP is 70-90%.DNMP in conjunction with chemical reaction, can be separately converted to corresponding D NTP after going on foot through DNMP enzyme reaction one, DNTP is through purification by chromatography, with the form precipitate and separate of sodium salt or barium salt.The DNTP purity of utilizing this method to obtain is 95%-97%.Can satisfy DNA synthetic needs.With DNA is that raw material calculates, and the total recovery of DNTP is 45-80%.The present invention not only makes simple that the production technique of DNTP becomes, and makes turnout reach the mole level, be applicable to industrial mass production, and contaminative is minimum.

Below in conjunction with embodiment, the present invention is described as follows:

Embodiment 1

The smart DNA of 100 gram commodity whales are dispersed in and make concentration in the water is 1% aqueous dna.DNA also can come from salmon essence, Shandong milt, beef liver, ox thymus gland etc. respectively.In dna solution, add zinc chloride 1.5 grams.Add deoxyribonuclease (550 units per ml) 250 milliliters.Enzymolysis carries out under 30-50 ℃.Enzymolysis time 10 hours.The adularescent precipitation generates in the enzymolysis process, and after enzymolysis was finished, freeze overnight removed by filter precipitation.The supernatant liquor chromatography separates.Chromatography condition: chromatograph post 150 * 1000mm, 100-200 order anionite-exchange resin (Cl ^-), desorbed solution 0.01N hydrochloric acid, flow velocity 200-250 ml/min.The DNMP evaporation concentration that obtains with the sodium-salt form precipitation, is precipitated as white powder, DAMP23.3 gram, DCMP26.5 gram, DGMP22.1 gram, DTMP18.8 gram, total recovery 90.7%.

The preparation of DNMP enzyme: microbial culture yeast extract 10%, peptone 0.5%, potassium primary phosphate 4%, dipotassium hydrogen phosphate 4%, glucose 10%, urea 3%, calcium chloride 0.2%, zinc chloride 0.2%, surplus is the aqueous solution.Substratum is inoculated after the steam sterilizing sterilization of 1.2 kg/cm, and inoculum size is 20-30%, and 50 ℃ of following violent stirring were cultivated 24 hours, centrifugal collection thalline under 1 ℃.Thalline is after high-speed stirring, with the Sodium phosphate dibasic of PH2.8--and the citrate buffer solution extraction, extraction liquid is not purified can directly to be used.

20 gram (about 6mmol) DNMP are dissolved in 500 ml waters, and it is 4.2-4.4 that solution is regulated PH with the potassium hydroxide solution of 10M, add Sodium phosphate dibasic 1.45 grams (10mmol), add 30 milligrams in zinc sulfate, ATP50 milligram again, add 300 DNMP of unit enzymes.50-55 ℃ is incubated 72 hours.After reaction finishes, heat to 90 ℃ 10 minutes, freezing, the centrifugal precipitation of removing.Supernatant liquor evaporation concentration to 200 milliliter, standby.

In the above-mentioned solution that obtains, add isopyknic ethanol, the ortho-phosphoric acid of 2.5 times of amounts, the DCC of 20 times of amounts, 80-90 ℃ was reacted 5 hours down.Reaction removes by filter precipitation after finishing, and evaporation removes and desolvates, and residue is dissolved in 10 premium on currency, chromatographic separation.Chromatography condition: chromatograph post 100 * 1000mm, 200-400 order anionite-exchange resin (Cl ^-), desorbed solution 0.1N hydrochloric acid, flow velocity 50-80 ml/min.The DNTP evaporation concentration that obtains with sodium-salt form precipitation, is precipitated as white powder, the DATP25.5 gram, and the DCMP24.1 gram, the DGMP20.7 gram, the DTMP26.2 gram, this step yield is about 67%, is starting raw material calculating with DNA, total recovery 60%.

Embodiment 2

The chromatographic separation of the preparation of DNMP, DNMP and DNTP is with embodiment 1.

The preparation of DNMP enzyme: microbial culture yeast extract 5%, peptone 2%, potassium primary phosphate 2%, dipotassium hydrogen phosphate 2%, glucose 10%, urea 1%, calcium chloride 0.2%, zinc chloride 0.2%, surplus is the aqueous solution.Substratum is inoculated after the steam sterilizing sterilization of 1.2 kg/cm, and inoculum size is 20-30%, and 50 ℃ of following violent stirring were cultivated 12 hours, centrifugal collection thalline under 1 ℃.Thalline is after high-speed stirring, with the Sodium phosphate dibasic of PH4.9--and the potassium phosphate buffer extraction, extraction liquid is not purified can directly to be used.

20 gram (about 6mmol) DNMP are dissolved in 500 ml waters, and it is 5.5-5.8 that solution is regulated PH with the potassium hydroxide solution of 10M, add Disodium phosphocreatine 3.27 grams (10mmol), add 50 milligrams in calcium chloride, ATP100 milligram again, add 220 DNMP of unit enzymes.50-55 ℃ is incubated 48 hours.After reaction finishes, heat to 90 ℃ 10 minutes, freezing, the centrifugal precipitation of removing.Supernatant liquor evaporation concentration to 200 milliliter, standby.

In the above-mentioned solution that obtains, add isopyknic ethanol, the ortho-phosphoric acid of 3 times of amounts, the DCC of 25 times of amounts, 80-90 ℃ was reacted 5 hours down.Reaction removes by filter precipitation after finishing, and evaporation removes and desolvates, and residue is dissolved in 10 premium on currency, chromatographic separation.Final DATP27.4 gram, the DCMP29.1 gram, the DGMP24.4 gram, the DTMP22.8 gram, this step yield is about 72%, is starting raw material calculating with DNA, total recovery 65%.

Embodiment 3

The preparation of DNMP enzyme is with embodiment 2.

20 gram (about 6mmol) DNMP are dissolved in 500 ml waters, and it is 6.5-6.8 that solution is regulated PH with the potassium hydroxide solution of 10M, add Disodium phosphocreatine 4.91 grams (15mmol), add 40 milligrams in magnesium chloride, ATP100 milligram again, add 250 DNMP of unit enzymes.50-55 ℃ is incubated 48 hours.After reaction finishes, heat to 90 ℃ 10 minutes, freezing, the centrifugal precipitation of removing.Supernatant liquor evaporation concentration to 200 milliliter, standby.

In the above-mentioned solution that obtains, add isopyknic ethanol, the ortho-phosphoric acid of 4 times of amounts, the DCC of 35 times of amounts, 80-90 ℃ was reacted 8 hours down.Reaction removes by filter precipitation after finishing, and evaporation removes and desolvates, and residue is dissolved in 10 premium on currency, chromatographic separation.Final DATP29.6 gram, the DCMP32.4 gram, the DGMP25.2 gram, the DTMP28.7 gram, this step yield is about 80.5%, is starting raw material calculating with DNA, total recovery about 72.5%.

Claims

1, a kind of preparation method of deoxyribonucleoside triphosphate may further comprise the steps:

(1) enzymolysis:

The microbial culture that the basic composition is 5-10% yeast extract of the substratum of DNMP enzyme preparation, the peptone of 0.5-2%, the potassium primary phosphate of 4-6%, the dipotassium hydrogen phosphate of 2-5%, the glucose of 10-15%, the urea of 0.5-3%, 0.2% calcium chloride, 0.2% zinc chloride, surplus is the aqueous solution;

The substratum of this DNMP enzyme preparation is inoculated after the steam sterilizing sterilization of 1.2 kg/cm, and inoculum size is 20-30%, and 50 ℃ of following violent stirring were cultivated 10-30 hour, centrifugal collection thalline under 1 ℃; Thalline is after high-speed stirring, with the damping fluid extraction of PH2-7; Damping fluid is Trisodium Citrate-sodium hydroxide-hydrochloric acid, Repone K-hydrochloric acid, acetate-sodium acetate, citric acid-sodium citrate, Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic-potassium primary phosphate, potassium primary phosphate-sodium hydroxide, Sodium phosphate dibasic-citric acid or Potassium Hydrogen Phthalate-sodium hydroxide solution; The not purified direct use of this extraction liquid;

Commodity DNA is dispersed in makes aqueous dna in the water, in described dna solution, add zine ion and make its concentration reach 2 * 10 ^-2-1 * 10 ^-4M, the amount that adds deoxyribonuclease according to the activity of deoxyribonuclease is 20 to 1/50th of a dna solution amount., enzymolysis carries out under 30-55 ℃; Enzymolysis 5-15 hour;

(2) enzymatic:

It is 4-8 that the DNMP aqueous solution is regulated PH with the potassium hydroxide solution of 10M, adds phosphoric acid salt, adds metal ion again, makes catalyzer with ATP, and according to the activity adding DNMP enzyme of DNMP enzyme, 50-55 ℃ is incubated 48-96 hour;

(3) chemosynthesis:

After enzymatic reaction is finished, add the alcoholic solvent of equivalent, the DCC that phosphoric acid that 2-5 doubly measures or phosphoric acid salt, 20-40 are doubly measured, 80-90 ℃ was reacted 4-8 hour down; The gained mixed solution is a phosphoric acid salt, DNMP, DNDP, DNTP and polyphosphide; This solution obtains purified DNTP through chromatographic separation.

2, the preparation method of deoxyribonucleoside triphosphate as claimed in claim 1 is characterized in that: phosphoric acid salt is Disodium phosphocreatine, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC or PEP; Metal ion is zinc sulfate, zinc chloride, zinc acetate, calcium chloride, sal epsom, magnesium chloride or bariumchloride.

3, the preparation method of deoxyribonucleoside triphosphate as claimed in claim 1 is characterized in that: alcoholic solvent is methyl alcohol, ethanol, butanols or butyl cellosolve.